Stöcklin R, Rose K, Green B N, Offord R E
Département de Biochimie Médicale, Centre Médical Universitaire, Geneva, Switzerland.
Protein Eng. 1994 Feb;7(2):285-9. doi: 10.1093/protein/7.2.285.
Insulin analogues labelled with stable isotopes (e.g. deuterium, 18O, 15N, etc.) are authentic (the native structure is rigorously maintained), non-radioactive (preferred for injection into man) and can easily be distinguished from endogenous insulin by mass spectrometry by virtue of their molecular masses. Appropriate combinations of amino-protecting groups (methylsulphonylethyloxycarbonyl and t-butoxy carbonyl), Edman degradation and chemical coupling were used to produce [octadeutero-PheB1]-porcine insulin and [octadeutero-PheB1-octadeutero-ValB2]-porcine insulin. The analogues were characterized by electrospray ionization mass spectrometry. Standard mixtures of labelled and unlabelled insulins were successfully studied by mass spectrometry. Isotope dilution mass spectrometry could therefore provide a useful direct measure of insulin under true physiological conditions, without many of the drawbacks of existing methods. In this regard, the analogue with 16 deuteriums was more suitable than the octadeuterated analogue, since the greater mass difference between the labelled and unlabelled forms enabled a lower mass spectrometric resolution to be used, resulting in higher sensitivity.
用稳定同位素（如氘、¹⁸O、¹⁵N等）标记的胰岛素类似物是真实的（严格保持天然结构），无放射性（适合注射入人体），并且通过质谱分析，凭借其分子量能够轻松与内源性胰岛素区分开来。使用氨基保护基团（甲基磺酰基乙氧基羰基和叔丁氧基羰基）、埃德曼降解和化学偶联的适当组合来制备[十八氘代 - PheB¹]-猪胰岛素和[十八氘代 - PheB¹ - 十八氘代 - ValB²]-猪胰岛素。通过电喷雾电离质谱对这些类似物进行表征。标记胰岛素和未标记胰岛素的标准混合物通过质谱分析得到成功研究。因此，同位素稀释质谱分析法能够在真实生理条件下提供一种有用的直接测量胰岛素的方法，而没有现有方法的许多缺点。在这方面，含16个氘的类似物比十八氘代类似物更合适，因为标记形式和未标记形式之间更大的质量差异使得可以使用较低的质谱分辨率，从而提高了灵敏度。
